Homeostasis of ion distributions across cell membranes is critical for the functional operation of living cells. Ion channels help transport ions into and out of the cell, and thus play important roles in many physiological functions. To accomplish the task, ion channels consist of pores, and the opening and closing of the pore, a process called gating, determines if ions can go through the channel. Our long-term interest is to understand how ion channels can function as nano-machines for ion transports, and how the gating functions of these nano-machines control cellular physiology. In this application I propose to study CLC channels, whose malfunctions result in various human diseases. Specifically, we will examine the mechanisms of the slow/common-gating of two voltage-gated CLC channels, CLC-0 and CLC-1. These two channels are highly homologous to each other-both channels are composed of two identical subunits, and each subunit forms a Cl--conducting pore. Two types of gating mechanisms are present in these channels-the fast- and the slow/common-gating. The fast-gating, a better-characterized mechanism, is known to control the opening and closing of the individual protopore. On the other hand, the mechanism of the slow/common-gating is poorly understood. Recently, we found that the slow/common-gating of CLC channels may involve a movement of the C-terminal cytoplasmic region of the channel. We also found that ATP, a physiologically critical molecule in all cells, inhibits CLC-1 by modulating the common-gating, presumably by binding to an ATP-binding site in the C- terminal cytoplasmic region. These preliminary results suggested a hypothesis that modulating the interaction between the two subunits may be the underlying mechanism of the ATP effect on the slow/common-gating. Physiologically, the inhibition of CLC-1's common-gating by ATP is critical for overcoming muscle fatigue. The slow/common-gating mechanism is also pathologically important because mutations that disrupt the common- gating of CLC-1 are known to cause the dominant form of myotonia. In this application we will combine electrophysiological and fluorescence imaging approaches to study how ATP binding exerts an inhibitory effect on CLC-1, and how myotonia mutations will disrupt the molecular function of CLC channels. These studies will unveil at the molecular level the roles of CLC-1 in muscle fatigue, and will also provide insight for the molecular mechanisms of CLC channelopathy. PUBLIC HEALTH RELEVANCE: This application will study CLC channels, which are chloride channels critical for chloride ion transports across cell membranes. In particular, the slow/common-gating mechanisms of CLC-0 and CLC-1 will be studied, and the regulation of the common-gating mechanism of CLC-1 by intracellular ATP and oxidants will be examined. Illustrating the underlying mechanisms of the interactions of the channel with these physiological ligands will further our understanding of the molecular functions of CLC-1 channels in muscle physiology and may help develop therapeutic strategies in treating diseases, such as myotonia, resulting from CLC channel defects.